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Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx™ Forensic Genomics System.
Forensic Sci Int Genet. 2017 11; 31:135-148.FS

Abstract

The ForenSeq™ DNA Signature Prep Kit (ForenSeq Kit) is designed to detect more than 200 forensically relevant markers in a single reaction on the MiSeq FGx™ Forensic Genomics System (MiSeq FGx System), including Amelogenin, 27 autosomal short tandem repeats (A-STRs), 7 X chromosomal STRs (X-STRs), 24 Y chromosomal STRs (Y-STRs) and 94 identity-informative single nucleotide polymorphisms (iSNPs) with the option to contain 22 phenotypic-informative SNPs (pSNPs) and 56 ancestry-informative SNPs (aSNPs). In this study, we evaluated the MiSeq FGx System on three major parts: methodological optimization (DNA extraction, sample quantification, library normalization, diluted libraries concentration, and sample-to-cell arrangement), massively parallel sequencing (MPS) performance (depth of coverage, sequence coverage ratio, and allele coverage ratio), and ForenSeq Kit characteristics (repeatability and concordance, sensitivity, mixture, stability and case-type samples). Results showed that quantitative polymerase chain reaction (qPCR)-based sample quantification and library normalization and the appropriate number of pooled libraries and concentration of diluted libraries provided a greater level of MPS performance and repeatability. Repeatable and concordant genotypes were obtained by the ForenSeq Kit. Full profiles were obtained from ≥100pg input DNA for STRs and ≥200pg for SNPs. A sample with ≥5% minor contributors was considered as a mixture by imbalanced allele coverage ratio distribution, and full profiles from minor contributors were easily detected between 9:1 and 1:9 mixtures with known reference profiles. The ForenSeq Kit tolerated considerable concentrations of inhibitors like ≤200μM hematin and ≤50μg/ml humic acid, and >56% STR profiles and >88% SNP profiles were obtained from ≥200-bp degraded samples. Also, it was adapted to case-type samples. As a whole, the ForenSeq Kit is a well-performed, robust, reliable, reproducible and highly informative assay, and it can fully meet requirements for human identification. Further, sensitive QC indicator and automated sample comparison function in the ForenSeq™ Universal Analysis Software are quite helpful, so that we can concentrate on questionable genotypes and avoid tedious and time-consuming labor to maximum the time spent in data analysis.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Guo F
Department of Forensic Medicine, Criminal Investigation Police University of China, No. 83, Tawan Street, Huanggu District, Shenyang 110854, Liaoning, PR China. Electronic address: nevergfaye@gmail.com.
Yu J
Criminal Science and Technology Institute of Liaoning Province, No. 2, Qishan Middle Road, Huanggu District, Shenyang 110032, Liaoning, PR China.
Zhang L
Department of Forensic Medicine, Criminal Investigation Police University of China, No. 83, Tawan Street, Huanggu District, Shenyang 110854, Liaoning, PR China.
Li J
Department of Forensic Medicine, Criminal Investigation Police University of China, No. 83, Tawan Street, Huanggu District, Shenyang 110854, Liaoning, PR China.

MeSH

DNA FingerprintingGene FrequencyHigh-Throughput Nucleotide SequencingHumansMicrosatellite RepeatsPolymerase Chain ReactionPolymorphism, Single NucleotideReproducibility of ResultsSequence Analysis, DNA

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

28938154

Citation

Guo, Fei, et al. "Massively Parallel Sequencing of Forensic STRs and SNPs Using the Illumina® ForenSeq™ DNA Signature Prep Kit On the MiSeq FGx™ Forensic Genomics System." Forensic Science International. Genetics, vol. 31, 2017, pp. 135-148.
Guo F, Yu J, Zhang L, et al. Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx™ Forensic Genomics System. Forensic Sci Int Genet. 2017;31:135-148.
Guo, F., Yu, J., Zhang, L., & Li, J. (2017). Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx™ Forensic Genomics System. Forensic Science International. Genetics, 31, 135-148. https://doi.org/10.1016/j.fsigen.2017.09.003
Guo F, et al. Massively Parallel Sequencing of Forensic STRs and SNPs Using the Illumina® ForenSeq™ DNA Signature Prep Kit On the MiSeq FGx™ Forensic Genomics System. Forensic Sci Int Genet. 2017;31:135-148. PubMed PMID: 28938154.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx™ Forensic Genomics System. AU - Guo,Fei, AU - Yu,Jiao, AU - Zhang,Lu, AU - Li,Jun, Y1 - 2017/09/08/ PY - 2017/02/12/received PY - 2017/08/06/revised PY - 2017/09/06/accepted PY - 2017/9/25/pubmed PY - 2017/12/12/medline PY - 2017/9/23/entrez KW - Evaluation KW - ForenSeq™ DNA Signature Prep Kit KW - Massively parallel sequencing (MPS) KW - MiSeq FGx(TM) Forensic Genomics System KW - Short tandem repeat (STR) KW - Single nucleotide polymorphism (SNP) SP - 135 EP - 148 JF - Forensic science international. Genetics JO - Forensic Sci Int Genet VL - 31 N2 - The ForenSeq™ DNA Signature Prep Kit (ForenSeq Kit) is designed to detect more than 200 forensically relevant markers in a single reaction on the MiSeq FGx™ Forensic Genomics System (MiSeq FGx System), including Amelogenin, 27 autosomal short tandem repeats (A-STRs), 7 X chromosomal STRs (X-STRs), 24 Y chromosomal STRs (Y-STRs) and 94 identity-informative single nucleotide polymorphisms (iSNPs) with the option to contain 22 phenotypic-informative SNPs (pSNPs) and 56 ancestry-informative SNPs (aSNPs). In this study, we evaluated the MiSeq FGx System on three major parts: methodological optimization (DNA extraction, sample quantification, library normalization, diluted libraries concentration, and sample-to-cell arrangement), massively parallel sequencing (MPS) performance (depth of coverage, sequence coverage ratio, and allele coverage ratio), and ForenSeq Kit characteristics (repeatability and concordance, sensitivity, mixture, stability and case-type samples). Results showed that quantitative polymerase chain reaction (qPCR)-based sample quantification and library normalization and the appropriate number of pooled libraries and concentration of diluted libraries provided a greater level of MPS performance and repeatability. Repeatable and concordant genotypes were obtained by the ForenSeq Kit. Full profiles were obtained from ≥100pg input DNA for STRs and ≥200pg for SNPs. A sample with ≥5% minor contributors was considered as a mixture by imbalanced allele coverage ratio distribution, and full profiles from minor contributors were easily detected between 9:1 and 1:9 mixtures with known reference profiles. The ForenSeq Kit tolerated considerable concentrations of inhibitors like ≤200μM hematin and ≤50μg/ml humic acid, and >56% STR profiles and >88% SNP profiles were obtained from ≥200-bp degraded samples. Also, it was adapted to case-type samples. As a whole, the ForenSeq Kit is a well-performed, robust, reliable, reproducible and highly informative assay, and it can fully meet requirements for human identification. Further, sensitive QC indicator and automated sample comparison function in the ForenSeq™ Universal Analysis Software are quite helpful, so that we can concentrate on questionable genotypes and avoid tedious and time-consuming labor to maximum the time spent in data analysis. SN - 1878-0326 UR - https://www.unboundmedicine.com/medline/citation/28938154/Massively_parallel_sequencing_of_forensic_STRs_and_SNPs_using_the_Illumina®_ForenSeq™_DNA_Signature_Prep_Kit_on_the_MiSeq_FGx™_Forensic_Genomics_System_ DB - PRIME DP - Unbound Medicine ER -